The goal of this proposal is to develop microdialysis sampling coupled to liquid chromatography as a biosensor capable of continuously monitoring biochemical processes in vivo with minimal perturbation of the biological system. Microdialysis sampling is consists of a short length of dialysis tubing (ca. 200 mum OD) through which solution is slowly pumped. Small molecules in the sample diffuse into the probe and are transported to a collection vial, while larger molecules are excluded by the membrane. Four applications of microdialysis will be investigated during this project. First, microdialysis techniques will be developed for pharmacokinetic studies. By implanting several probes into a test animal the pharmacokinetics of a drug at several sites will be simultaneously determined. For example, probes will be implanted in the liver, the muscle, and intravenously to study the relationship of blood levels to systemic distribution and hepatic metabolism. Second, microdialysis will be used to study the interaction of drugs with blood proteins. Only the free fraction of the drug is sampled by microdialysis because protein bound drug can not diffuse through the dialysis membrane. Third, multiple probes will also be used to study transport of drugs across the blood-brain barrier. Finally, using probes implanted subcutaneously, transdermal drug delivery will be investigated. The project will also encompass fundamental aspects of mass transport across membranes under microdialysis conditions. The quality of the analytical data depends on an understanding of these transmembrane processes. Parameters of the analyte, the membrane, the probe design and the sample matrix will be investigated. The effect of properties such as charge, hydrophobicity and molecular weight on transport will be determined. Other parameters, such as sample viscosity and ionic strength, temperature gradients across the membrane, and dialysis tubing length and diameter will also be studied. Finally, the microdialysis analysis system will be automated. A specially designed dual injection valve will be used for continuous sample collection and injection. This will relieve the experimenter from sample handling tasks so that total attention can be focused on the biological aspects of the experiment. To further enhance the resolution of the experiment, microdialysis will be directly coupled to mass spectrometry. The microdialysis probe will provide a sample amenable to direct injection into the mass spectrometer which will provide the selectivity to determine drugs and their metabolites.